Hemoglobin (Hb) variants - Structure and Functions
- 1. Hb variants Dr. Shanmugha Priya R.A
- 2. Objectives: • Haemoglobin - Genetics, structure, Function • Hb variants - Types, Genetics, laboratory testing • Transfusion Therapy
- 3. Introduction: Haemoglobin • Iron-containing substance in RBCs that transports oxygen from the lungs to tissues and transports carbon dioxide back to lungs • 250 million molecules of Hb per RBC. Each Hb binds four oxygen molecules • Has an oxygen-binding capacity of 1.34 mL O2 per gram, which increases the total blood oxygen capacity seventy-fold compared to dissolved oxygen in blood.
- 4. Haemoglobin Genetics : • More than one Hb gene exists. MC: Hemoglobin A - coded by genes HBA1, HBA2 & HBB. • Mutations in the genes for the Hb protein result in haemoglobin variants. • At least six genes that control globin synthesis in humans, resulting in the formation of six structurally different polypeptide chains that are designated α, β, γ, δ, ξ, and ς chains.
- 6. • All normal and most abnormal hemoglobin molecules are tetramers consisting of two different pairs of polypeptide chains, each chain forming a monomeric subunit and each with a heme group. • Heme group is composed of a porphyrin ring which contains an iron (Fe) atom in its center which binds to O2 • The blood of a normal adult human contains at least six different species of hemoglobin molecules, all of which have the same principal structure and function. Haemoglobin structure:
- 7. Haemoglobin Structure and Function
- 8. R form Vs T form
- 9. Haemoglobin Variants • Are part of the normal embryonic and foetal development. • >1000 Hb variants are known to exist. • These are mainly missense mutations that destabilize Hb, alter Hb-O affinity or most commonly alter Hb function minimally. • Deletions, multiple aminoacid substitutions, antitermination mutations, altered post translational processing also can cause Hb variants .
- 10. Haemoglobin Variants - types • Mutant Non –Pathologic Variants- do not cause disease, may be adaptive, are rarely life threatening or health compromising. • Mutant Pathologic variants – cause a group of hereditary diseases termed the Hemoglobinopathies. All these diseases produce Anemia .
- 11. Study of Hb variants- Uses • Identification of Hb gene mutations as a cause for cyanosis, erythrocytosis, or mild hemolysis in otherwise healthy patients provides reassurance and minimizes additional diagnostic procedures, sparing expense and risk. • Efforts to understand how Hb variants produce their structural, biochemical, and clinical effects has generated important insights into red blood cell function and also created general paradigms for the study of protein biology.
- 12. Types of Hb Variants In the embryo • Gower 1 (ζ2ε2) • Gower 2 (α2ε2) • Hemoglobin Portland I (ζ2γ2) • Hemoglobin Portland II (ζ2β2) In the fetus • Hemoglobin F (α2γ2) .
- 13. Types of Hb Variants After birth: • Hemoglobin A (α2β2) – The most common with a normal amount over 95% • Hemoglobin A2 (α2δ2) – δ chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5–3.5% • Hemoglobin F (α2γ2) – In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. However, the level of Hb F can be elevated in persons with sickle-cell disease and beta-thalassemia.
- 14. Pathological Hb Variants • Hemoglobin D-Punjab – (α2βD 2) • Hemoglobin H (β4) –formed by a tetramer of β chains. Present in variants of α thalassemia. • Hemoglobin Barts (γ4) –formed by a tetramer of γ chains. Present in variants of α thalassemia. • Hemoglobin S (α2βS 2) – A variant form of haemoglobin found in people with sickle cell disease. There is a variation in the β-chain gene, causing a change in the properties of hemoglobin, which results in sickling of red blood cells.
- 15. Pathological Hb Variants • Hemoglobin C (α2βC 2) – due to a variation in the β-chain gene. causes a mild chronic hemolytic anemia. • Hemoglobin E (α2βE 2) –due to a variation in the β-chain gene. causes a mild chronic hemolytic anemia. • Hemoglobin AS – A heterozygous form causing sickle cell trait with one adult gene and one sickle cell disease gene • Hemoglobin SC disease – A compound heterozygous form with one sickle gene and another encoding Haemoglobin C.
- 16. Prevalence of haemoglobinopathies - worldwide
- 17. Unstable variant • Causes congenital heinz body haemolytic anaemia • Genetic mutations alters steps in globin processing resulting in destabilisation of Hb • Mutations at the α1β1 interface can cause hemolytic anemia by inhibiting heterodimer formation, favoring the accumulation of free globin subunits, which themselves are unstable, particularly α chains
- 19. High Oxygen Affinity Variants • Hemoglobin variants with increased O2 affinity cause erythrocytosis by stimulating erythropoietic drive • commonly results from amino acid substitutions that stabilize the R (high O2 affinity) state relative to the T (low O2 affinity) state and/or inhibit responses to environmental allosteric regulators that stimulate O2 release, including H+ (Bohr effect) or 2,3DPG • high affinity variants frequently result from substitutions that alter α1β2 interface
- 22. Low Oxygen Affinity Variants • Low O2 affinity Hb variants typically present with cyanosis. • commonly results from globin amino acid substitutions that tip the quaternary equilibrium of Hb tetramers from R (high O2 affinity) state relative to the T (low O2 affinity) state • Some low affinity hb variants are unstable and therefore causes not only cyanosis but also heinz body hemolytic anaemia
- 23. Methaemoglobin (M- type) variants • Hemoglobin iron must be in its reduced (Fe2+ , ferrous) state to bind O2. Moreover, oxidized (Fe3+ , ferric, met) Hb is intrinsically unstable with a tendency to release heme. • Presents clinically as pseudocyanosis • In vitro analyses of the red cell and isolated Hb samples can usually distinguish wild-type metHb resulting from toxins or defective reductase systems and M-type Hb variants that are predisposed to spontaneous oxidation
- 24. Globin Chain Elongation Mutants • Antitermination and frameshift mutations that add irrelevant amino acids to the carboxyl terminus of globin proteins produce interesting variants that can damage erythrocytes • Hb Constant Spring contributes to α -thalassemia syndromes, particularly when combined with two a- globin deletional alleles (–/aCSa), which produces a severe form of HbH disease
- 26. Variants that Affect Multiple Hemoglobin Functions • Amino acid substitutions within critical regions of globin proteins can produce multiple effects. • Mutations that alter the heme pocket commonly produce multiple biochemical effects.
- 28. Indications for lab testing
- 29. • confirmation or exclusion of the presence of a structural variant, thalassaemia trait or both. • If a structural haemoglobin variant is present - ascertain the clinical significance of the particular variant so that the patient is appropriately managed. • If thalassaemia trait is present- usually not necessary to determine the precise mutation present because the clinical significance is usually negligible.( exception - antenatal patient whose partner has also been found to have thalassaemia trait.) Investigation of patients with a suspected haemoglobinopathy
- 30. • If prenatal diagnosis is done - mutation analysis to predict fetal risk accurately must be undertaken • Defined protocol should be devised to suit individual local requirements. • The data obtained from the clinical findings, blood picture and electrophoresis or HPLC will usually indicate in which direction to proceed.
- 31. 1. Blood count and film examination 2. Collection of blood and preparation of haemolysates 3. Cellulose acetate electrophoresis, Tris buffer, pH 8.5 4. Citrate agar or acid agarose gel electrophoresis, pH 6.0 5. Automated HPLC 6. IEF 7. Tests for Hb S 8. Detection of unstable haemoglobins 9. Detection of Hb Ms 10. Detection of altered affinity haemoglobins 11. Differentiation of common structural variants 12. Neonatal screening 13. Tests, such as zinc protoporphyrin estimation, to exclude iron deficiency as a cause of microcytosis 14. Molecular techniques 15. Procedures for use in under- resourced laboratories Laboratory detection of Hb variants
- 32. Suggested scheme of investigation for structural variants
- 33. • Isoelectric focusing or high-performance liquid chromatography (HPLC) - most commonly used tests, identify most structurally abnormal Hbs. In this way, many benign Hb variants are discovered incidentally. • Specific laboratory tests a. Physical methods of Hb separation b. Hb-O2 binding curve c. Visible wavelength spectroscopy d. Hemoglobin stability testing e. Specialized testing
- 34. Physical methods of Hb separation • Electrophoretic and chromatographic techniques examine the physical properties of α1β1 dimers or individual globin subunits. • Controls used - HbA, HbS, HbC, HbF, HbA. • Hb variants show altered migration in these assays. • Cellulose acetate and citrate agar electrophoresis - most commonly used. • Isoelectric focusing and HPLC- more sensitive.
- 35. Hb-O2 binding curve • Indicates the percent (%) oxygenated Hb at a given O2 partial pressure • Hemoglobin variants with an abnormally high O2 affinity produce a “left-shifted” equilibrium curve, whereas low O2 affinity variants will cause “right shift.” • Determination of Hb-O2 affinity responses to allosteric regulators 2,3DPG or pH changes (H+ ) - structural causes of the observed phenotypes. • Only a few clinical laboratories currently offer this assay.
- 36. Visible wavelength spectroscopy • Hemoglobin variants with amino acid substitutions in the heme pocket affect visible light absorbance. • Pulse oximetry is a non invasive spectrophotometric test. But it gives confusing results with variant Hbs showing unique light absorbance properties. • In these cases, analysis of arterial blood O2 concentration may be required to rule out hypoxia.
- 37. Hemoglobin stability testing • Hb stability is impaired in variants that are associated with hemolytic anemia. • Hemoglobin stability tests measure the propensity for Hb to denature on exposure to various stresses including heat, isopropanol, mechanical agitation and zinc acetate. • Heinz body test uses supravital stains (methylene blue or crystal violet) to detect aggregated globins within erythrocytes
- 38. Specialized testing • Mass spectrometry analysis (hemolysate) and DNA sequencing of globin genes - identify amino acid and nucleotide alterations associated with Hb variants • Crystallographic analysis is the highest resolution approach to determine the effects of globin amino acid substitutions on molecular structure.
- 39. Transfusion Therapy in sickle cell disease
- 40. •When anemia is the predominant concern - transfusion goals such as adequate hemoglobin concentration are appropriate. •For complications in which sickle cell vasoocclusion is the overriding concern, the primary objective of RBC transfusion is to achieve a favorable balance between the relative concentrations of normal and sickle red cells. (HbS concentration < 30%) Transfusion Therapy in sickle cell disease
- 41. Transfusion related complication 1. Alloimmunisation – • 20 – 30% • Common in patients receiving multiple transfusion • Multiple alloantibodies • Hyperresponders, non responders 2.Delayed haemolytic transfusion reaction • Blood from Sickle Cell Trait Donors for Patients without Sickle Cell Disease/ with sickle cell disease
- 42. Transfusion therapy- Thalassemia • “Hypertransfusion regimen” - hemoglobin levels usually maintained higher than 9.5 or 10 g/dL; consists of RBC transfusions of about 20 mL/kg, given whenever the patient’s hemoglobin falls below the targeted level • Transfusion regimen for Cooley anemia that maintains the hematocrit above 35%, but this “supertransfusion” approach is no longer recommended • Iron chelation
- 43. Summary • Mutations in the genes for the Hb protein result in haemoglobin variants • To date , > 1000 Hb variants are found • Identification and study of Hb variants minimizes additional diagnostic procedures, sparing expense and risk and giving insight into some of the important red cell functions • Not all Hb variants are harmful, some of them give a survival advantage • Appropriate transfusion goal should be achieved while transfusing the patients with pathological Hb variants for better prognosis.
- 44. References: • Boundless. “Red Blood Cells.” Boundless Biology. Boundless, 26 May. 2016. Retrieved 19 Oct. 2016 from https://www.boundless.com/biology/ textbooks/boundless - biology-textbook/the-circulatory-system-40/components-of-the- blood-225/red-blood-cells-847-12092/ • https://www.ncbi.nlm.nih.gov/books/NBK54103 • World wide distribution picture-By Armando Moreno Vranich - Original work of Armando Moreno Vranich, Public Domain, https://commons wikimedia.org/w/index.php?curid=11568835 • Globin Gene Server; Hardison et al. 2002; Giardine et al. 2011 • Hemoglobin Variants: Biochemical Properties and Clinical Correlates .Christopher S. Thom, Claire F. Dickson,David A. Gell, Mitchell J. WeisCold Spring Harb Perspect Med 2013;3:a011858 • Rossi’s Priciple of transfusion medicine, 4 th edition /
Editor's Notes
- #3: = each red blood cell carries one billion molecules of oxygen The affinity of hemoglobin for oxygen is less than its structural analog myoglobin There are approximately 25 trillion red blood cells in the five liters of blood in the human body, which could carry up to 25 sextillion (25 × 1021) molecules of oxygen at any time. In mammals, the lack of organelles in erythrocytes leaves more room for the hemoglobin molecules. The lack of mitochondria also prevents use of the oxygen for metabolic respiration. Only mammals have anucleated red blood cell except camel
- #7: Hemoglobin A (A for adult) makes up 92% of the total hemoglobin concentration in a normal adult human.
- #10: Variations in hemoglobin amino acid sequences, as with other proteins, may be adaptive. For example, hemoglobin has been found to adapt in different ways to high altitudes. fetal hemoglobin has a noticeably higher affinity for oxygen than does maternal hemoglobin.
- #23: “pseudocyanosis,” (i.e., low Hb-O2 saturation), despite adequate arterial oxygenation.
- #35: Hemoglobin variants with an abnormally high O2 affinity will become saturated at lower pressures producing a “left-shifted” equilibrium curve, whereas mutations that reduce will cause the opposite “right shift.”
- #36: For example, M-type Hbs show characteristic spectra that can distinguish them from methemoglobinemia caused by an enzyme deficiency in the metHb reductase system (Bunn and Forget 1986; Dailey and Meissner 2012; Ganz and Nemeth 2012; Schechter 2012). Pulse oximetry is a non invasive spectrophotometric test that measures absorbance ratios at specific wavelengths for oxygenated (660 nm) and deoxygenated (940 nm) blood
- #38: DNA sequencing - elucidates the presence of an Hb variant., But additional biochemical and structural studies are required to determine how the variant affects Hb function
- #41: the majority of these are anti- Rh(E). Anti-Kell (K) and anti-Kidd (Jka and Jkb) account for approximately 20% of alloantibodies in transfused patients with SCD.87 Lewis antibodies account for up to one-third of alloantibodies,